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. 2005 Aug;25(16):7239-48.
doi: 10.1128/MCB.25.16.7239-7248.2005.

Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization

Yoshiaki Kamada  1 , Yuko Fujioka, Nobuo N Suzuki, Fuyuhiko Inagaki, Stephan Wullschleger, Robbie Loewith, Michael N Hall, Yoshinori Ohsumi
Affiliations

Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization

Yoshiaki Kamada et al. Mol Cell Biol. 2005 Aug.

Abstract

The target of rapamycin (TOR) protein kinases, Tor1 and Tor2, form two distinct complexes (TOR complex 1 and 2) in the yeast Saccharomyces cerevisiae. TOR complex 2 (TORC2) contains Tor2 but not Tor1 and controls polarity of the actin cytoskeleton via the Rho1/Pkc1/MAPK cell integrity cascade. Substrates of TORC2 and how TORC2 regulates the cell integrity pathway are not well understood. Screening for multicopy suppressors of tor2, we obtained a plasmid expressing an N-terminally truncated Ypk2 protein kinase. This truncation appears to partially disrupt an autoinhibitory domain in Ypk2, and a point mutation in this region (Ypk2(D239A)) conferred upon full-length Ypk2 the ability to rescue growth of cells compromised in TORC2, but not TORC1, function. YPK2(D239A) also suppressed the lethality of tor2Delta cells, suggesting that Ypks play an essential role in TORC2 signaling. Ypk2 is phosphorylated directly by Tor2 in vitro, and Ypk2 activity is largely reduced in tor2Delta cells. In contrast, Ypk2(D239A) has increased and TOR2-independent activity in vivo. Thus, we propose that Ypk protein kinases are direct and essential targets of TORC2, coupling TORC2 to the cell integrity cascade.

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Figures

FIG. 1.
FIG. 1.
N-truncated Ypk2 suppresses tor2 mutants. A. YYK241 (ade2 ade3 tor2 p414GAL1[TOR2 ADE3]) was transformed with YEp352 (vector), YEp352[full-length YPK2] (YPK2FL), YEp352[N-truncated YPK2] (YPK2-224), and YEp352[N-truncated ypk2-K373A (kinase dead)] (YPK2-224KD), as indicated. The transformants were grown on YEPD plates for 4 days at 30°C. Sectoring colonies showed independence of TOR2 plasmid, i.e., suppression of tor2 mutation. B. Alignment of the amino acid sequences around the M224 of Ypk2. Ypk1, Ypk2 (S. cerevisiae), and Gad8 (S. pombe) are shown. The arrowhead highlights M224 of Ypk2, and asterisks denote the conserved region shared among these protein kinases. C. YPK2 genes (cloned into YEp352) harboring a point mutation in the conserved region as indicated (right panel) were transformed into YYK241, and the resultant transformants were grown on YEPD plates for 2 days at 30°C (left) or 37°C (right). Note that these YPK2 genes expressed full-length of Ypk2 protein under the control of their own promoters.
FIG. 2.
FIG. 2.
YPK2D239A restores Mpk1 activation and suppresses actin cytoskeleton organization defects in tor2 mutant cells. A. Wild-type cells (YMW1) and tor2 mutant cells (YYK241) harboring indicated YPK2 plasmids were transformed with pRS424[HA-tagged MPK1 (MPK1HA)] to express Mpk1HA protein. Cells were grown at 37°C, and phosphorylation of Mpk1HA was determined by immunoblotting with anti-diphospho-p44/p42 MAPK (Cell Signaling Technology) (top panel). Total Mpk1 was determined by immunoblotting with anti-HA antibody (bottom panel). B. Wild-type (SH100) and tor2-21 mutant (SH121) cells harboring YEp352[YPK2D239A] grown in YEPD at 30°C were shifted to 37°C for 6 h. The actin cytoskeleton was stained with rhodamine-phalloidin as described in Materials and Methods. The percentage of cells exhibiting a polarized actin patch is also shown (bottom).
FIG. 3.
FIG. 3.
Tor2 directly phosphorylates Ypk2 in vitro. A. Detection of immunoprecipitated HA-tagged Tor2 (HATOR2). HATor2wild type (WT; lanes 1 and 2) or HATor2D2298E (kinase dead mutant [KD]; lanes 3 and 4) were immunoprecipitated in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of anti-HA antibody. Immunocomplexes were subjected to 7% SDS-PAGE gels, and HATor2 protein was detected by immunoblotting with anti-HA antibody. B. In vitro Tor2 kinase assay. Immunoprecipitated HATor2 (wild type [WT] or kinase dead [KD]) was incubated with [γ-32P]ATP and 4E-BP (lanes 1 to 4 and lanes 9 to 12) or recombinant Ypk2 (lanes 5 to 8 and lanes 13 to 16) at 30°C for 60 min and stopped with sample buffer, and proteins were separated by SDS-PAGE. The results of Coomassie staining (left) and autoradiography (kinase assay) (right) are shown. * and **, migration of heavy and light chains of the immunoprecipitation antibody. C. Tor2 phosphorylates Ypk2 at the turn motif and the hydrophobic motifs. In vitro kinase assays were carried out using the wild type (WT; lanes 1, 2, and 4) and the turn- and hydrophobic-motif mutants (S641A T659A, S641A, and T659A; lanes 3, 5, and 6, respectively) of Ypk2.
FIG. 4.
FIG. 4.
YPK2D239A overcomes mutation in cis, Tor2-dependent phosphorylation sites. A. Mutation sites of Ypk2. D239 is in a “TOS-like” conserved region, and S641 and T659 are in the turn motif and the hydrophobic motif (sites phosphorylated by Tor2), respectively. B. (Top) YPK2 genes (cloned into YEp352) harboring mutation at D239 or/and Tor2-phosphorylation sites as indicated (right) were transformed into YYK373 (ypksΔ p415GAL1[YPK2-224]). The transformants were grown on YEP galactose (Ypk2-224 expressed; left) or YEPD (Ypk2-224 not expressed; center) plates for 2 days at 30°C.(Bottom) Serial dilutions of the indicated transformants were spotted on SCgalactose-URA (left) and SCglucose-URA (right) and grown for 2 days at 30°C. C. A temperature-sensitive tor2-21 (SH121 [TOR1 tor2-21]) mutant was transformed with YEp352-based YPK2 plasmids as indicated (right) and grown on YEPD for 2 days at 30°C (left) or 36.5°C (center). Wild-type control cells (SH100 [TOR1 TOR2]) are also shown. D. YPK2 mutant genes were transformed into YYK353 (TOR1 tor2Δ pRS316[TOR2]) (top panel) and YYK357 (tor1Δ tor2Δ pRS316[TOR2]) (bottom panel) as indicated in the right panel, and the resultant transformants were grown on YEPD (left) and 5-fluoro-orotic acid (5FOA) plate (center) for 2 days at 30°C. On 5FOA plates, cells must lose the pRS316[TOR2] (harboring URA3 marker) plasmid to grow (19).
FIG. 5.
FIG. 5.
YPK2D239A suppresses growth defects associated with TORC2 disruption but not TORC1 disruption. GAL1 promoter-KOG1 (a component of TORC1; RL93a)- and GAL1 promoter-AVO1 (a component of TORC2; RL23-1c)-containing cells harboring YPK2 plasmids as indicated (right) were grown in SCgalactose glycerol-Ura (left) or SCglucose-Ura (center).
FIG. 6.
FIG. 6.
YPK2D239A acts as a tor2-specific suppressor. A. (Top) YPK2 alleles (cloned into YEp352) harboring mutations at D239 or/and T501 (Pkh phosphorylation site) were transformed into YYK373 (ypksΔ p415GAL1[YPK2-224]). The transformants were grown on YEPgalactose (Ypk2-224 expressed; left) or YEPD (Ypk2-224 not expressed; center) plates for 2 days at 30°C. (Bottom) A temperature-sensitive tor2-21 mutant (SH121 [TOR1 tor2-21]) was transformed with YEp352-based YPK2 plasmids as indicated (right) and grown on YEPD for 2 days at 30°C (left) or 36.5°C (center). Wild-type control cells (SH100 [TOR1 TOR2]) are also shown. B. Temperature-sensitive pkh mutant cells (INA106-3B [pkh1D398G pkh2Δ]) and wild-type control cells (INA28-1B [PKH1 pkh2Δ]) were transformed with YEp352-based YPK2 plasmids as indicated (right) and grown on YEPD for 2 days at 30°C (left) or 36.5°C (center).
FIG. 7.
FIG. 7.
Protein kinase activity of Ypk2 is regulated by TOR2. A. Wild-type (SH100) and tor2-21 mutant (SH121) cells harboring YEp352-based HA-tagged YPK2 plasmids as indicated were grown in YEPD at 30°C. Ypk2HA was immunoprecipitated from cell lysates and subjected to in vitro kinase assays (Kinase assay; top panel). Immunoprecipitated Ypk2HA was monitored by immunoblot (IB; bottom). Radioactivity and intensity of immunoblot signal were measured by BAS2000 and LAS1000 (Fuji Film), respectively, and specific activity of each sample was calculated (shown in the figure). B. Ypk2 kinase activity was assessed as described above. A kinase dead Ypk2 mutant (K373A) displayed no detectable protein kinase activity (lanes 3 and 5).
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